1. Field of the Invention
The present invention is directed to a method for selectively blocking glucocorticoid receptors by contacting the receptors with a glucocorticoid receptor blocking effective amount of arsenite or methyl methanethiolsulfonate (MMTS) to a sample.
2. Description of Related Art
A large number of responses to steroid hormones are mediated by the cognate receptor protein. In the absence of a functional, wild-type receptor, these responses to steroid hormones cannot be observed. For example, lymphocytes and leukemic cells are normally killed by glucocorticoids and many breast cancer cells are killed by estrogens; but, cell toxicity is not observed in leukemic cells lacking glucocorticoid receptors or in breast cancer cells lacking estrogen receptors. Similarly, patients resistant to vitamin D were found to have a point mutation in that region of the receptor protein that interacts with the DNA of regulated genes. Furthermore, the amount of functional receptor protein has been found to be important both for the level of hormone response and, when the target cell contains more than one type of receptor, for the type of receptor-mediated response which is observed. Thus quantitation of the amount of functional receptor in a given cell or tissue is crucial for predicting whether a steroid-induced response is possible and what the magnitude of that response will be.
The four current methods for detecting the presence of receptors vary in their ability to detect functional receptors. Receptor mRNA levels can be determined by quantitative Northern blots since the cDNAs of all of the steroid receptors have now been cloned (see Evans, Science, 240, 889-895, 1988 and references therein). However, this method cannot distinguish a priori between the mRNAs of functional and non-functional receptor proteins (Northrop et al., J. Biol. Chem., 261, 1106-11070, 1986) and cannot guarantee the presence of receptor protein. Quantitative Western blots with anti-receptor antibodies cannot distinguish between functional and non-functional proteins (Martin et al., Proc. Natl. Acad. Sci., 85, 2533-2537, 1988). Binding assays, typically with radiolabelled (e.g., .sup.3 H and .sup.125 I) steroids or ligands, do detect and can be used to determine the binding parameters of biologically active receptors. Other methods of labelling the ligand, such as, for example, with fluorescent groups or with biotin that can be detected by fluorescent spectroscopy or avidin-linked enzyme assays respectively, can also be employed for use in these binding assays. Unfortunately, since the expression of steroid-regulated responses requires many steps that are distal to the binding of steroid to receptor, a lesion in any of these steps can also lead to steroid resistance. Thus, an assay which measures the desired biological response is the most accurate method for determining whether a given cell will respond to steroids. However, measurements of the final response (such as cell death) are often technically very difficult or time consuming. In the treatment of breast cancer, one compromise in this direction has been to determine the presence of "functional" estrogen receptors by measuring the amount of progesterone receptors, which are induced by estrogens (Clark et al., N. Engl. J. Med., 309, 1343-1347, 1983). However, in most other cases, people have settled for the simple assay of steroid binding activity in order to predict the quantity of "functional" receptors.
Initially it was thought that each steroid hormone might bind exclusively to its cognate receptor. This hypothesis has since been found to be incorrect. In fact, it appears to be impossible to find any steroid that will bind to just one type of receptor. Cross-reactivity is especially high with glucocorticoid, progesterone, and mineralocorticoid receptors, which is probably related to the high degree of homology that is seen in the steroid-binding domains of these three receptors (Evans, 1988, supra). Thus various expensive synthetic steroids are required to selectively bind to, or block, one receptor in those many tissues that are known to contain two of these receptors. However, when all three receptors are present in the same tissue, as is the case for pituitary and mammary tissue, it can be very difficult to quantitate the binding to just one receptor.
A different approach to eliminating the problem of cross-reactivity in steroid binding would be to find an inexpensive reagent that would selectively recognize a structural element of the steroid-binding domain that is different for each receptor. The present invention was discovered with the above disadvantages and concerns in mind.